The aim of this research program is to investigate the anti-hemostatic and anti-inflammatory compounds in the saliva of blood-feeding insects and ticks which allow efficient blood feeding and enhancement of pathogen transmission. Anti-hemostatic compounds of interest include anti-clotting, anti- platelet and vasodilators. Anti-inflammatory compounds include immunomodulatory compounds as well as compounds that modify effector arms of the immune response, such as anti-complement activity found in the saliva of some ticks, sand flies and mosquitoes. While the vector attempts to modify the feeding site to enhance success of blood feeding, such site becomes locally compromised in its ability to react to injury and becomes an easier site for pathogen invasion. On the other hand, when the vertebrate mounts an immune response to these salivary compounds, pathogen transmission may be compromised. Novel pharmaceuticals and novel targets for vaccine development will be ultimate benefits of this program. The work involves obtaining saliva or salivary glands of the insects and ticks under study, bioassay of their biological activity using both in vivo and in vitro tests, purification of the individual activities using chromatographic and electrophoretic techniques, microsequencing of the peptides and proteins, and final identification of the total primary sequence by molecular biology methods. Confirmation of the clones is found by expression in bacterial or eukaryotic cell lines. We have initiated a reverse approach where salivary cDNA libraries from blood-feeding insects and ticks are being mass sequenced and expressed recombinantly, providing new insights in the discovery of novel compounds. Summary Vector arthropods, such as mosquitoes, triatomine bugs and ticks, salivate while they puncture our skin in their search of blood. This saliva contains dozens to hundreds of compounds that have anti-clotting, anti-platelet, vasodilatory, anti-inflammatory, and immunomodulatory functions. While helping the vector to feed, it also modifies the site where pathogens are injected and, in many cases, facilitates the infection process. For this reason, salivary proteins of vectors can be used as vaccine targets for the diseases they transmit. Salivary proteins can also be used as immuno-epidemiological markers of vector exposure, and in themselves can have potent and novel pharmacological activities. Because the saliva of hematophagous animals is under attack of their host's immune system, their constituents are under a rapid evolutionary pressure in an arms' race scenario with their hosts, causing an enormous variety of unique protein families even in closely related organisms. The section of vector biology aims at uncovering the biodiversity of salivary proteins in the near 500 genera of blood sucking arthropods, and to discover the function of the novel protein families that we encounter. Accordingly, we have developed a two pronged approach focusing in sialotranscriptome discovery projects and functional sialomic studies. In addition to these core studies on the saliva of vector arthropods, the section also collaborated with other members of the LMVR, and other extramural scientists lending its expertise in bioinformatics. In the current fiscal year, members of the Section of Vector Biology contributed to a total of 7 papers as detailed below: Sialotranscriptome discovery and evolution projects: Because host hemostasis (the physiological process that prevents blood loss, consisting of platelet aggregation, blood clotting and vasoconstriction) is a complex and redundant phenomenon, the salivary glands of blood sucking arthropods consist of a magic potion with diverse chemicals that in a redundant way counteract host mechanisms to prevent blood loss, allowing the fast acquisition of a meal. Salivary transcriptome made in the past few years indicate that the magic potion consists of 70-100 different proteins in the case of mosquitoes, for example, to over 1,000 in the case of ticks (Ticks feed for several days and have to disarm host immune reactions, in addition to the hemostatic system). Transcriptome studies also show that the salivary proteins of blood sucking arthropods are at a very fast pace of evolution, perhaps explaining why every genus studied so far has several unique protein families. Indeed, there are unique proteins found at the subgenus level. Given we can now describe in detail the sialotranscriptome (from the Greek word sialo = saliva) of a single organism, we can ask now what the universe of salivary proteins is associated to blood feeding, the so called sialoverse. There are near 19,000 species of blood sucking arthropods in 500 different genera. If we find (minimally) 5 novel protein families per genus (within the 70-500 proteins in each sialome), there are at least 2,500 novel proteins to be discovered, each one with an interesting pharmacological property. We have so far explored less than 20 genera of blood sucking arthropods, and it is our goal to extend sialotranscriptome discovery to map this pharmacological mine for future studies, and in the process learn the paths taken by genomes in their evolution to blood feeding and identify proteins with pharmacological and vaccine potential. Following up on our previous findings of transcripts derived from viral genomes within salivary transcriptomes, a novelty this past year was our collaboration with the Argentinian virologist, Dr. Humberto Debat, to better characterize these viral transcripts, thus characterizing organ viromes. Transcriptome and virome studies: In the current fiscal year, we produced two papers related to sialotranscriptome and evolution of hematophagous arthropods, including the first time disclosure of the sialome of Amazonian anophelines and their viromes (1), as well as the sialome and the virome of the biting fly, Haematobia irritans (2), By assembly of publicly available transcriptome reads of anopheline mosquitoes, we discovered a mosquito virus in Anopheles epiroticus that was previously only found in Culex mosquitoes (3). From the analysis of sialotranscriptome of ticks infected or not infected with Rickettsia organisms, we found expression of antimicrobial microplusin peptides in infected ticks (4). Functional studies: We advanced our knowledge regarding the structure, function and evolution of vector salivary proteins by analyzing the structure of the D7 family of proteins that are overexpressed in the salivary glands of mosquitoes and sand flies. Because these two groups of insects evolved independently to blood-feeding, this scenario of convergent evolution offers a natural replicate of the evolutionary process toward the perfect salivary potion (5), We also described the salivary protein families of hematophagous insects in one review article (6). Bioinformatic collaborations: Dr. Ribeiro collaborated with intra and extra mural investigators lending his expertise in bioinformatics. With Dr. Louis Miller from LMVR he helped to analyze transcriptomes of Plasmodium vivax infecting susceptible and non-susceptible monkeys, identifying candidates of parasite proteins associated with erythrocyte invasion (7).